ProjectSummary Currentlyapprovedimmunotherapieshaveshownefficacyinpatientswithimmunogenictumortypes, suchasmelanomaandnon-smallcelllungcancer.However,immunotherapiesarelesseffectivefortumors withlowlevelsofinfiltratinglymphocytesandalowmutationrate,limitingthenumberofprimedTcellswithin thetumormicroenvironment.OurlaboratoryhasdevelopedaDNAvaccineplatformusingsyntheticconsensus sequencestobreaktolerancetogermlineantigens.Thesevaccinescanbreaktolerancetonativeproteinsin mousemodelsandinduceanti-tumoractivity.However,DNAvaccinesarestilllimitedbytheimmune suppressivemicroenvironment,whichpreventsCD8Tcellinfiltrationandactivationwithinthetumor. Specifically,theextracellularmatrixglycosaminoglycanhigh-molecular-weight(hmw)hyaluronan(HA)is abundantinthetumormicroenvironmentandisknowntorestrictTcellactivationinother(non-tumor)contexts throughengagementofCD44.HyaluronidaseisanenzymethatconvertshmwHAtolow-molecular-weight (lmw)HA,whichisknowntoengagetheinnateimmunesystemthroughToll-likeRecptors(TLRs). Hyaluronidasetreatmentofpancreaticandprostatecancerswasalsoshowntoenhanceangiogenesisand improvechemotherapeuticdelivery.Wethereforehypothesizethathyaluronidasetreatmentinthetumor microenvironmentwillenhanceTcellinfiltrationandactivationintumorsresultinginimproved efficacywhencombinedwithDNAvaccineimmunotherapyinapancreaticcancermodel. Weshowinpreliminaryexperimentsthatcombinationtherapywithhyaluronidaseandvaccinationina prostatecancermodelinducedcompletetumorregressionsinoverhalfofthemice,whiletreatmentwitheither vaccinealoneorhyaluronidasealonedidnotinduceanycompleteregressions.Ourfirstaimistocharacterize changestothetumormicroenvironmentuponhyaluronidasetreatmentincombinationwithourDNAvaccines, andtodeterminethespecificcontributionsofhmwandlmwHAtothesephenotypes.Oursecondaimisto determinethemechanismofactionofthiscombinationtherapyusingfunctionalcellulardepletionexperiments, andagonisticorblockingantibodiestoHAreceptors.